It is possible through the techniques of genetic engineering to cause a host cell to create "heterologous" polypeptides, i.e., polypeptides which are not naturally created by that species of cell. A variety of mammalian polypeptides have been produced in E. coli cells, such as somatostatin, described by Itakura et al, Science 198: 105 (1977); the component A and B chains of human insulin, disclosed by Goeddel et al, Proc. Nat'l. Acad. Sci. USA 76: 106 (1979); human growth hormone, disclosed by Goeddel et al, Nature 281: 544 (1979); human leukocyte interferon, disclosed by Goeddel et al, Nature 287: 411 (1980); human fibroblast interferon, disclosed by Goeddel et al Nucleic Acids Res. 8 (18): 4057 (1980); and human serum albumin, as disclosed by Lawn et al, Nucleic Acids Res. 9(22): 6103 (1981).
In order to cause a host cell to express a heterologous polypeptide, a structural sequence (also commonly called a coding sequence) which codes for a polypeptide is usually placed near a promoter sequence which causes the structural sequence to be transcribed into messenger RNA (mRNA). A variety of promoter sequences have been used to promote the expression of chimeric genes in E. coli, such as the lac promoter, Backman and Ptashne, Cell 13: 65 (1978); the trp promoter, Hallewell and Emtage, Gene 9: 27 (1980); and the phage lambda P.sub.L promoter, Bernard et al, Gene 5: 59 (1979).
A variety of gene promoter/operator systems are "inducible"; their activity can be varied substantially by the presence or absence of a certain substance or condition. For example, the lac promoter system of E. coli has a relatively low level of activity in the absence of lactose, a particular type of sugar molecule. However, in the presence of lactose (i.e., when lactose is added to the culture medium which contains the E. coli cells), the lac promoter system becomes much more active, and causes a higher level of transcription of the DNA sequence near the promoter. See, e.g., J. Miller and W. Reznikoff, The Operon, 2nd edition, Cold Spring Harbor Labs, N.Y. (1982). Other inducible promoter systems include the trp promoter (which has relatively low activity if an excess of tryptophan is present, and higher activity if a low concentration of tryptophan is present or if 3 B-indolylacrylic acid is present; see Hallewell and Emtage, supra) and the phage lambda promoter (which has a relatively low level of activity at 37.degree. C. and higher activity at 42.degree. C. in the presence of a temperature-sensitive mutant lambda repressor; see Bernard, supra).
The recA gene of E. coli has been cloned and its nucleotide sequence has been reported; see T. Horii et al, "Organization of the recA gene of E. coli," P.N.A.S. USA 77:313 (1980) and A. Sancar et al, "Sequences of the recA gene and protein," P.N.A.S. USA 77:2611 (1980). The recA polypeptide is involved in a variety of functions, including genetic recombination and a class of functions called "SOS" functions which occur when the cell is placed under certain types of stress. The recA polypeptide is commercially available from P-L Biochemicals, Inc. (Milwaukee, Wis.). Expression of the recA gene can be induced by a variety of substances including nalidixic acid and mitomycin C, and by various stress conditions such as ultraviolet radiation. See E. M. Witkin, "Ultraviolet mutagenesis and inducible DNA repair in E. coli," Bacteriol. Rev. 40:869 (1976). If appropriate mutant host cells are used, then certain other stress conditions, such as elevated temperature or thymine starvation, may be used to induce expression of the recA polypeptide.
Prior to the filing of the parent application in this case, there had been two reports of the use of a recA promoter in a man-made chimeric gene. The first report, T. Miki et al, "Construction of a Fused Operon Consisting of the recA and kan (Kanamycin Resistance) Genes and Regulation of Its Expression by the lexA Gene," Mol. Gen. Genet. 183: 25-31 (1981), discussed the use of a recA promoter to promote transcription of a structural sequence which was translated into the enzyme, kanamycin phosphotransferase (KPT). KPT inactivates kanamycin, an antibiotic which is toxic to E. coli cells. KPT is naturally present in various strains of E. coli which contain certain plasmids or transposons; in the presence of kanamycin, the KPT gene can serve as a selectable marker. The apparent purpose of the work reported by Miki, et al was to evaluate the interrelationship between the recA gene and a different gene, designated the lexA gene, which produces a protein called lexA which represses transcription of the recA gene.
The second report, S. Casaregola et al, "Quantitative Evaluation of recA Gene Expression in E. coli," Mol. Gen. Genet. 185: 430-439 (1982), involved a recA::lac chimeric gene. Since the different enzymatic activities of the recA protein are difficult to measure, Casaregola et al created a chimeric gene comprising a structural sequence which is translated into beta-galactosidase (B-gal), controlled by a recA promoter. B-gal is an enzyme that is naturally present in various strains of E. coli.
In addition, one report was published in 1983 after the parent application was filed. This report, by S. I. Feinstein et al, Nucleic Acids Research 11(9): 2927-2941 (1983), describes the use of the recA promoter to express human interferon genes in E. coli.
As used herein, the term "endogenous polypeptide" refers to a polypeptide that exists naturally in a certain genus or species of host cells. For example, some naturally occurring strains of E. coli contain an enzyme, having kananycin phosphotransferase (KPT). Therefore, even though not all strains of E. coli contain KPT, it may be regarded as endogenous with respect to cells of the species E. coli. By comparison, the term "heterologous polypeptide", as used herein, refers to a polypeptide that does not naturally exist in a genus or species of host cells. For example, a mammalian or plant polypeptide would be regarded as heterologous with respect to E. coli and other bacterial cells.
It is normally much easier to cause the expression by a chimeric gene of an endogenous polypeptide in a certain type of bacterial host cell (such as KPT or B-galactosidase, both of which naturally exist in some strains of E. coli cells) than to cause a bacterial host cell to express a heterologous polypeptide, such as interferon or somatostatin. This is due to a variety of complex processes which are not completely understood. Some of the factors which are believed to impede the expression or accumulation of a heterologous polypeptide in a host cell include the following:
1. the heterologous polypeptide may be degraded by the host cell.
2. the heterologous polypeptide may have toxic effects upon the host cell, presumably because of interaction with one or more naturally occurring proteins or other substances which are essential to the functioning of the cell.
The problem of degradation is believed to be more severe with regard to relatively small heterologous polypeptides than with regard to large heterologous polypeptides. Although a wide variety of heterologous polypeptides with more than about 100 amino acid residues have been expressed in E. coli, relatively few heterologous polypeptides with less than about 50 amino acid residues have been expressed in E. coli, despite numerous attempts to do so.
A number of efforts to overcome the problems of causing heterologous polypeptides to be expressed in bacterial cells have utilized "fusion" polypeptides. Such fusion polypeptide normally utilize a portion of a naturally occurring E. coli polypeptide (often called a "carrier" polypeptide) coupled to a polypeptide selected by the investigator. Such fusion polypeptides are usually created by inserting a heterologous structural sequence into the structural sequence of a selected E. coli gene. The inserted structural sequence is transcribed into chimeric mRNA under the control of the promoter/operator sequence of the E. coli gene, and the chimeric mRNA is translated into a polypeptide under the control of the 5' non-translated region and the AUG start codon of the E. coli gene. In a fusion polypeptide, the inserted structural sequence must be in the proper reading frame relative to the AUG start codon of the E. coli gene. See e.g., Goeddel et al P.N.A.S. (1979), supra, and U.K. Patent Application GB No. 2,007,676A (Itakura and Riggs, publ. 1979).
Despite these and other technological advances, various problems continue to limit the range or utility of efforts to use E. coli cells to express certain heterologous polypeptides, such as somatostatin. Such problems include:
1. expression of the heterologous polypeptide even while the promoter is repressed. This tends to slow down or divert other metabolic and reproductive processes which are more useful during the growth phase. This reduces the number or vitality of host cells that can be grown in a certain amount of time, thereby reducing the amount of heterologous polypeptide which can be expressed and accumulated by the cells during a time-limited fermentation cycle.
2. lack of adequate flexibility in the control of a promoter/operator system. Most known promoter/operator systems can be induced effectively by only one or a few known substances or conditions. However, any particular inducing substance or condition may have other deleterious effects upon a particular strain of host cells or a particular metabolic reaction. It would be preferable to have a promoter system which can be induced by a wide variety of substances or conditions.
3. for reasons which are not yet understood, certain heterologous polypeptides have apparent detrimental effects upon certain cells. This is usually manifested by several effects, such as:
a. difficulties encountered by researchers who are attempting to isolate cells containing plasmids that would be expected to result from specific DNA manipulative efforts, and
b. plasmid instability. For example, Itakura et al report that the plasmids they created which contain a chimeric B-gal/somatostatin gene was relatively unstable; see Itakura et al, Science 198: 1056 at 1062 (1977). A relatively minor degree of plasmid instability, expressed over a large number of generations, can lead to a very large subpopulation which does not express the desired polypeptide, if the absence of the polypeptide confers a reproductive advantage upon the subpopulation.
Although Itakura et al, supra, have reported the expression of a fusion polypeptide containing somatostatin coupled to part of a B-gal polypeptide from E. coli, their paper discusses several limitations on the efficient expression of that fusion polypeptide in E. coli, including plasmid instability and low levels of accumulation within the host cells. Efforts by Monsanto scientists to create similar plasmids have confirmed that similar problems do (described below) do in fact prevent the accumulation of desirable levels of B-gal/somatostatin fusion polypeptides in E. coli.